Thermostable DNA polymerases used for PCR are those belonging to family A and those belonging to family B. Examples of known DNA polymerases belonging to family A include DNA polymerase derived from Thermus thermophilus (Tth polymerase), DNA polymerase derived from Thermus aquaticus (Taq polymerase), and the like. Examples of known DNA polymerases belonging to family B include DNA polymerase derived from Pyrococcus furiosus (Pfu polymerase), thermostable DNA polymerase derived from Thermococcus litoralis (Tli polymerase), DNA polymerase derived from Thermococcus kodakaraensis (KOD DNA polymerase), and the like.
DNA polymerases belonging to family A have been heretofore used as thermostable DNA polymerases used for PCR for reasons such as good amplification efficiency and easy condition setting. However, recent various investigations into reaction compositions etc. have enabled easy condition setting even with DNA polymerases belonging to family B. In addition, DNA polymerases belonging to family B have high fidelity and thermostability, and are resistant to inhibiting substances carried into the reaction system; therefore, DNA polymerases belonging to family B have been widely used not only in the research field, but also in the field of forensic medicine, such as in genetic diagnosis and clinical diagnosis. DNA polymerases belonging to family B have also been widely used in testing for microorganisms in foods and the environment, etc.
DNA polymerases belonging to family B are, however, problematic in that when dUTP is incorporated, reactions stop because of their high fidelity. Since dUTP is also generated by thermal decomposition of dCTP, DNA polymerases belonging to family B are affected by dUTP generated during thermal cycling of PCR, and thus cannot sufficiently exhibit their high amplification efficiency. In particular, DNA polymerases belonging to family B are known to have reduced amplification efficiency in PCR using a long-chain target as a template, which requires a long thermal cycling time, and in PCR performed at multiple cycles using a small amount of template.
Additionally, since PCR is a highly sensitive detection method, carryover of amplification products from previous PCR may lead to false-positive results. To address this problem, a technique is taken in which PCR is performed using substrates containing dUTP instead of dTTP to incorporate uracil bases into amplification products, and contamination (carryover) PCR amplification products are degraded by treating with uracil-N-glycosylase (UNG) when the next PCR is performed (dUTP/UNG decontamination method) (Non-patent Literature 1). Although DNA polymerases belonging to family B have advantages of high fidelity and thermostability as well as resistance to inhibiting substances carried into the reaction system, they cannot be used in such a technique, since dUTP cannot be incorporated (Non-patent Literature 2, Non-patent Literature 3, and Non-patent Literature 4).
In recent years, interaction of DNA polymerases belonging to family B with nucleic acid containing dUTP has been studied, and an analysis of crystal structure of DNA polymerase derived from Thermococcus gorgonarius (Tgo polymerase) with dUTP was carried out. The results of the analysis suggest the presence of uracil binding pockets that are formed by amino acids at positions 1 to 40 and amino acids at positions 78 to 130 in Tgo polymerase, and show that strong interaction of one of the uracil binding pockets with dUTP shops extension reactions of PCR (Non-patent Literature 5).
It was then found that DNA polymerases in which an amino acid associated with uracil binding pockets is modified have reduced affinity for dUTP, thus allowing PCR to be performed even in the presence of dUTP. However, it has been impossible for even these DNA polymerases, in which an amino acid associated with uracil binding pockets is modified, to provide sufficient amounts of PCR amplification and to fully exhibit amplification efficiency of DNA polymerases belonging to family B. In addition, since reduction in enzyme activity has been expected, multiple mutations in amino acids associated with uracil binding pockets have not heretofore been considered (Patent Literature 1 and 2).